This invention relates to obtaining purified bindweed and buckwheat extracts by collecting components of a homogenized aqueous solution of bindweed material. High molecular weight extracts of bindweed were previously shown to inhibit the growth of two different types of virulent tumors in mice. The extracts possessed immunopotentiating effects as evidenced by tumor infiltration by white blood cells in the tumors of treated animals, and induction of lymphocyte proliferation. They further possessed antiangiogenic properties as demonstrated in the chick chorioallantoic membrane assay. The method of extraction of the present bindweed and buckwheat extracts is very simple and requires a minimum of purification. The present bindweed and buckwheat extracts have utility as low-toxicity, anti-cancer drugs for human and animal use.
Recently the concept of using biological response modifiers (BRMs) has been considered for the treatment of cancer. Some BRMs are not directly cytotoxic to tumor cells, but possess qualities which change the environment of the organism. Immune stimulation and inhibition of new blood vessel growth are two biological response modification strategies with potential in the treatment of cancer. Examples of immune stimulators with known anti-tumor activity are Polysaccharide K, beta 1,3, glucan, and the Maruyama vaccine. All of which contain high-molecular weight polysaccharides and/or protein. Examples of angiogenesis inhibiting molecules include TMP-470, and angiostatin, which have demonstrated anti-tumor activity. While investigating extracts of field bindweed for anti-tumor activity, we initially tried to isolate low-molecular weight alkaloids, which are known to be toxic, and which we suspected of having traditional chemotherapeutic, or tumor-cytotoxic activity.
It was found that the low-molecular weight extracts, containing the toxic alkaloids, exhibited little anti-tumor activity, while high molecular weight extracts, which excluded the toxic alkaloids contained significant anti-tumor activity by acting as biological response modifiers.
One object of the invention is to provide a pharmaceutical consisting of high molecular weight extracts of field bindweed (Convolvulus arvensis) which have low toxicity to normal cells and induce anti-tumor effects in animals, inhibit the growth of blood vessels, and enhance immune function in mammals. Preferably these high molecular weight extracts have components less than about 500 Daltons removed. More preferably the removed components are less than about 1,000. Even more preferably, the removed components are less than about 3,000 Daltons. Even more preferably, the removed components are less than about 5000, more preferably 10,000.
One embodiment of the invention is to isolate the high molecular weight extracts of field bindweed (Convolvulus arvensis) using a molecular weight filter or alternatively precipitation with ammonium sulfate.
A further object of the present invention is to provide a method for isolating high molecular weight extracts of field bindweed (Convolvulus arvensis), which have low toxicity by virtue of removal of toxic low-molecular weight components of a crude extract. Preferably the components less than about 3000 Daltons are removed. More preferably the components less than about 6,500 Daltons are removed. Even more preferably, the components less than about 10,000 Daltons are removed.
One embodiment of the invention is to remove the low molecular weight extracts of field bindweed (Convolvulus arvensis) using a molecular weight filter or alternatively precipitation with ammonium sulfate.
A further object of the present invention is to provide a method for use of high molecular weight extracts of field bindweed (Convolvulus arvensis), which have low toxicity to normal cells and induce anti-tumor effects in animals.
A further object of the present invention is to provide a method for use of high molecular weight extracts of field bindweed (Convolvulus arvensis), which inhibit new blood vessel growth.
A further object of the present invention is to provide a method for use of high molecular weight extracts of field bindweed (Convolvulus arvensis), which exhibits immunoenhancing effects in animals.
A further object of the invention is a pharmaceutical composition for treating cancer, inhibiting the growth of new blood vessels, and/or enhancing immune function in a mammal, which is an effective amount of an aqueous extract of Convolvulus, and a pharmaceutically acceptable vehicle. Preferably the extract is prepared by homogenizing Convolvulus arvensis plant parts and preparing the aqueous extract from the plant parts. Preferably, the plant parts exclude seeds and flowers. Preferably, the extract is prepared using three volumes of water per volume of homogenized plant parts. Preferably, solids are removed from the extract. Preferably, the extract is lyophilized after removal of the solids.
A further object of the invention is a method for preparing non-toxic extracts of Convolvulus by preparing an aqueous extract of Convolvulus. Preferably, the solid components of the plant are removed.
A further object of the invention is a method for the treatment of cancer in a mammal by administering a pharmaceutical composition of Convolvulus in an amount effective to slow or stop the growth of said cancer.
A further object of the invention is a method for inhibiting blood vessel growth in a mammal by administering a pharmaceutical composition of Convolvulus in an amount effective to slow or stop the growth of said blood vessels.
A further object of the invention is a method for enhancing immune function in a mammal by administering the pharmaceutical composition of Convolvulus in an amount effective to enhance said immune function. Preferably, the immune function is selected from the group consisting of lymphocyte growth and phagocyte activity.
A further object of the invention is a pharmaceutical composition for treating cancer, inhibiting the growth of new blood vessels, and/or enhancing immune function in a mammal, which is an effective amount of an aqueous extract of Polygonum, and a pharmaceutically acceptable vehicle. Preferably the extract is prepared by homogenizing Polygonum plant parts; and preparing the aqueous extract from the plant parts. Preferably, the plant parts exclude seeds and flowers. Preferably, the extract is prepared using three volumes of water per volume of homogenized plant parts. Preferably, solids are removed from the extract. Preferably, the extract is lyophilized after removal of the solids.
A further object of the invention is a method for preparing non-toxic extracts of Polygonum by preparing an aqueous extract of Polygonum. Preferably, the solid components of the plant are removed.
A further object of the invention is a method for the treatment of cancer in a mammal by administering a pharmaceutical composition of Polygonum in an amount effective to slow or stop the growth of said cancer.
A further object of the invention is a method for inhibiting blood vessel growth in a mammal by administering a pharmaceutical composition of Polygonum in an amount effective to slow or stop the growth of said blood vessels.
A further object of the invention is a method for enhancing immune function in a mammal by administering the pharmaceutical composition of Polygonum in an amount effective to enhance said immune function. Preferably, the immune function is selected from the group consisting of lymphocyte growth and phagocyte activity.
We discovered a high molecular weight extract of bindweed that surprisingly has low toxicity to normal cells and induces anti-tumor effects, inhibits the growth of blood vessels, and enhances immune function in mammals. We also found that the low molecular weight components of bindweed extract contained alkyloids on the order of 300-500 Daltons that were toxic to both normal cells and tumor cells. In order to remove the toxic low molecular weight components we used a molecular weight sieve. In general, the method of the invention for obtaining high molecular weight extracts of Convolvulus arvensis consists of boiling fresh or aged bindweed in an aqueous solution to obtain a brown tea-like mixture. This mixture is then centrifuged or filtered to remove the solid material to create a solution. It is only necessary to remove the 300-500 molecular weight components to remove the toxicity to normal cells. However, we found that the low molecular weight components might nonspecifically stick to the membrane and come back off. Therefore, preferably, a larger molecular weight sieve is usedxe2x80x94on the order of 1000 Daltons, more preferably, 3000 Daltons, even more preferably 5000 Daltons. We find that even when molecular weight components of up to 10,000 Daltons are removed, the extract retains its anti-tumor effects in animals, its ability to inhibit the growth of blood vessels, and its ability to enhance immune function in mammals. In the Examples below, after boiling the fresh or aged bindweed in an aqueous solution to obtain a brown tea-like mixture, the solution is then passed through a molecular weight filtration device to obtain a high molecular weight retentate (BWR), or precipitated using ammonium sulfate to isolate a high molecular weight precipitate (BWP). Thereafter, the high molecular weight extract is lyophilized, or otherwise concentrated. The extract is subsequently assayed for anti-tumor, immunoenhancing, and anti-angiogenesis activity.
SDS-Page, IEF, and a protein assay, are then used in order to further characterize the components of the extract. The extract is also assayed for molecular weight using a Superose12-HR sizing column (Pharmacia Biotech) using known molecular weight standards.
Further features and advantages of the present invention will become apparent to those of skill in the art in view of the detailed description of the preferred embodiments which follows when considered together with the attached drawings and claims.
Although other material and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Example 1 describes the steps required to prepare the high molecular weight extracts of bindweed.